System and method of analysis of a protein using liquid chromatography-mass spectrometry

ABSTRACT

The present disclosure pertains to method and system of characterizing a protein using an electrospray ionization source.

FIELD

The present invention generally pertains to a method and system ofcharacterizing a protein.

BACKGROUND

Electrospray ionization (ESI)-mass spectrometry (MS) coupled tochromatographic and electrophoretic separation techniques is a keytechnology in proteomics. It has become an important tool for in-depthcharacterization of protein biopharmaceuticals in analytical labs tosupport their developmental and regulatory filings. Proteinbiopharmaceuticals must meet very high standards of purity and hence itis important to monitor and characterize proteins during differentstages of drug development and production.

Liquid chromatography-mass spectrometry (LC-MS)-based analysis ofprotein biopharmaceuticals could benefit tremendously from improved dataquality, which can subsequently lead to improved drug characterizationwith higher confidence and less ambiguity. To provide characterizationof different protein attributes, a wide variety of LC-MS-based assayscan be performed, within which peptide mapping analysis and intact massanalysis are most routinely and widely applied. To improve theconfidence of the analysis and reduce the ambiguity associated with datainterpretation, constant efforts need to be made to improve the dataquality from the LC-MS analysis, including using optimized experimentalprocedures, fine-tuned instrument parameters as well as more advancedmass spectrometers.

From the foregoing it will be appreciated that a need exists forimproved methods and systems to improve protein characterization.

SUMMARY

Growth in the development, manufacture and sale of protein-basedbiopharmaceutical products has led to an increasing demand for methodsand systems for characterizing a protein.

Embodiments disclosed herein satisfy the aforementioned demands byproviding methods and systems for the characterization of a protein.

The disclosure, at least in part, provides a method of characterizing aprotein in a sample, for example: supplying the sample to an inlet of anelectrospray ionization source, wherein the electrospray ionizationsource comprises a container having a cap with an inlet line port and anoutlet line port, a sheath gas inlet line, a modified desolvation gasoutlet line, an electrospray ionization probe with a sheath gas inlet;generating ions of components of the protein in the sample at an outletof the electrospray ionization source; and analyzing the ions using amass spectrometer to identify the components of the protein tocharacterize the protein.

In some exemplary embodiments, the method of characterizing a protein ina sample can comprise an electrospray ionization source comprising acontainer having a cap with an inlet line port and an outlet line portand a sheath gas inlet line capable of providing sheath gas to the inletline port.

In some exemplary embodiments, the method of characterizing a protein ina sample can comprise an electrospray ionization source comprising acontainer having a cap with an inlet line port and an outlet line portand a modified desolvation gas outlet line capable of connecting theoutlet line port to a sheath gas inlet of an electrospray ionizationprobe.

In some exemplary embodiments, the method of characterizing a protein ina sample can comprise an electrospray ionization source comprising acontainer surrounded by a second container.

In some exemplary embodiments, the method of characterizing a protein ina sample can comprise an electrospray ionization source with anelectrospray ionization probe in a positive polarity mode.

In some exemplary embodiments, the method of characterizing a protein ina sample can comprise an electrospray ionization source with anelectrospray ionization probe in a negative polarity mode.

In some exemplary embodiments, the method of characterizing a protein ina sample can comprise conducting an intact mass analysis.

In some exemplary embodiments, the method of characterizing a protein ina sample can comprise conducting a peptide mapping analysis.

In some exemplary embodiments, the method of characterizing a protein ina sample can comprise characterizing an antibody.

In some exemplary embodiments, the method of characterizing a protein ina sample can comprise an electrospray ionization source with a containerhaving an organic solvent.

In some exemplary embodiments, the method of characterizing a protein ina sample can comprise an electrospray ionization source with a containerhaving an organic solvent and an acid.

In some exemplary embodiments, the method of characterizing a protein ina sample can comprise an electrospray ionization source with a containerhaving an organic solvent and a base.

In some exemplary embodiments, the method of characterizing a protein ina sample can comprise an electrospray ionization source with a containerhaving acetonitrile.

In some exemplary embodiments, the method of characterizing a protein ina sample can comprise an electrospray ionization source with a containerhaving triethylamine (TEA).

In some exemplary embodiments, the method of characterizing a protein ina sample can comprise an electrospray ionization source with a containerhaving trifluroacetic acid.

In some exemplary embodiments, the method of characterizing a protein ina sample can comprise an electrospray ionization source having anelectrospray ionization probe with an auxiliary gas inlet.

In some exemplary embodiments, the method of characterizing a protein ina sample can comprise an electrospray ionization source having anelectrospray ionization probe with an electrospray emitter needle.

In some exemplary embodiments, the method of characterizing a protein ina sample can comprise an electrospray ionization source comprising acontainer having a cap with an inlet line port and an outlet line portand a sheath gas inlet line capable of providing nitrogen gas to theinlet line port.

In some exemplary embodiments, the method of characterizing a protein ina sample can comprise an electrospray ionization source having anelectrospray ionization probe with an auxiliary gas inlet, wherein theauxiliary gas inlet can be capable of being supplied with an auxiliarygas.

In some exemplary embodiments, the method of characterizing a protein ina sample can comprise an electrospray ionization source having anelectrospray ionization probe with an auxiliary gas inlet, wherein theauxiliary gas inlet can be capable of being supplied with nitrogen gas.

In some exemplary embodiments, the method of characterizing a protein ina sample can comprise an electrospray ionization source having anelectrospray ionization probe including an electrospray emitter needle,a sheath gas flow plumbing, and an auxiliary gas flow plumbing.

In some exemplary embodiments, the method of characterizing a protein ina sample can comprise an electrospray ionization source having anelectrospray ionization probe including an electrospray emitter needle,a sheath gas flow plumbing, and auxiliary gas flow plumbing, wherein theelectrospray ionization probe can be configured to direct flow in thesheath gas flow plumbing coaxially to the electrospray emitter needle.

In some exemplary embodiments, the method of characterizing a protein ina sample can comprise an electrospray ionization source having anelectrospray ionization probe including an electrospray emitter needle,a sheath gas flow plumbing, and an auxiliary gas flow plumbing, whereinthe electrospray ionization probe can be configured to direct flow inthe auxiliary gas flow plumbing coaxially to the electrospray emitterneedle.

In some exemplary embodiments, the method of characterizing a protein ina sample can comprise an electrospray ionization source having anelectrospray ionization probe, wherein the electrospray ionization probecan be a heated electrospray ionization probe.

In some exemplary embodiments, the method of characterizing a protein ina sample can comprise an electrospray ionization source having a capwith an inlet line port and a sheath gas inlet line, wherein the sheathgas inlet line can be partially inserted into the inlet line port.

In some exemplary embodiments, the method of characterizing a protein ina sample can comprise an electrospray ionization source having a capwith an outlet line port and a modified desolvation gas outlet line,wherein the a modified desolvation gas outlet line can be partiallyinserted into to the outlet line port.

In some exemplary embodiments, the method of characterizing a protein ina sample can comprise an electrospray ionization source having acontainer comprising a cap with an inlet line port and an outlet lineport, a sheath gas inlet line, and a modified desolvation gas outletline, wherein the electrospray ionization source can be configured toallow a flow of a sheath gas from the sheath gas inlet line through thecontainer into the desolvation gas outlet line.

In some exemplary embodiments, the method of characterizing a protein ina sample can comprise an electrospray ionization source having acontainer comprising a cap with an inlet line port and an outlet lineport, a sheath gas inlet line, and a modified desolvation gas outletline, wherein the electrospray ionization source can be configured toallow a flow of a sheath gas from the sheath gas inlet line through thecontainer having an organic solvent into the desolvation gas outletline.

In some exemplary embodiments, the method of characterizing a protein ina sample can comprise an electrospray ionization source having acontainer comprising a cap with an inlet line port and an outlet lineport, a sheath gas inlet line, and a modified desolvation gas outletline, wherein the electrospray ionization source can be configured toallow a flow of a sheath gas from the sheath gas inlet line through thecontainer having an organic solvent and an additional chemical componentinto the desolvation gas outlet line.

In some exemplary embodiments, the method of characterizing a protein ina sample can comprise an electrospray ionization source capable ofproviding an electrospray with a solvent flow rate of greater than about5 μL/min.

In some exemplary embodiments, the method of characterizing a protein ina sample can comprise an electrospray ionization source having acontainer, wherein the container can be a pressure resistant container.

In some exemplary embodiments, the method of characterizing a protein ina sample can comprise an electrospray ionization source capable of beingconnected to a liquid chromatographic system.

In some exemplary embodiments, the method of characterizing a protein ina sample can comprise characterizing a digestion product of a protein.

In some exemplary embodiments, the method of characterizing a protein ina sample can comprise analyzing the ions using a mass spectrometer toidentify the components of the protein to characterize the protein,wherein the mass spectrometer can be a tandem mass spectrometer.

This disclosure, at least in part, provides a liquid chromatography massspectrometry system, comprising a liquid chromatography device, anelectrospray ionization source having a container having a cap with aninlet line port and an outlet line port, a sheath gas inlet line, amodified desolvation gas outlet line, an electrospray ionization probeand a mass spectrometry device.

In some exemplary embodiments, the liquid chromatography massspectrometry system can comprise a electrospray ionization sourcecomprising a container having a cap with an inlet line port and anoutlet line port and a sheath gas inlet line capable of providing sheathgas to the inlet line port.

In some exemplary embodiments, the liquid chromatography massspectrometry system can comprise a electrospray ionization sourcecomprising a container having a cap with an inlet line port and anoutlet line port and a modified desolvation gas outlet line capable ofconnecting the outlet line port to a sheath gas inlet of an electrosprayionization probe.

In some exemplary embodiments, the liquid chromatography massspectrometry system can comprise an electrospray ionization probe thatcan be capable of being run in a positive polarity mode.

In some exemplary embodiments, the liquid chromatography massspectrometry system can comprise an electrospray ionization probe thatcan be capable of being run in a negative polarity mode.

In some exemplary embodiments, the liquid chromatography massspectrometry system can comprise an electrospray ionization source witha container capable of being filled with an organic solvent.

In some exemplary embodiments, the liquid chromatography massspectrometry system can comprise a electrospray ionization source with acontainer capable of being filled with an organic solvent and anadditional chemical component, wherein the additional chemical componentcan include an acid, base, salt, or combinations thereof.

In some exemplary embodiments, the liquid chromatography massspectrometry system can comprise an electrospray ionization source witha container capable of being filled with an organic solvent and an acid.

In some exemplary embodiments, the liquid chromatography massspectrometry system can comprise an electrospray ionization source witha container capable of being filled with an organic solvent and a base.

In some exemplary embodiments, the liquid chromatography massspectrometry system can comprise an electrospray ionization sourcehaving an electrospray ionization probe with an auxiliary gas inlet.

In some exemplary embodiments, the liquid chromatography massspectrometry system can comprise an electrospray ionization sourcehaving an electrospray ionization probe with an electrospray emitterneedle.

In some exemplary embodiments, the liquid chromatography massspectrometry system can comprise an electrospray ionization sourcecomprising a container having a cap with an inlet line port and anoutlet line port and a sheath gas inlet line capable of providingnitrogen gas to the inlet line port.

In some exemplary embodiments, the liquid chromatography massspectrometry system can comprise an electrospray ionization sourcehaving an electrospray ionization probe with an auxiliary gas inlet,wherein the auxiliary gas inlet can be capable of being supplied with anauxiliary gas.

In some exemplary embodiments, the liquid chromatography massspectrometry system can comprise an electrospray ionization sourcehaving an electrospray ionization probe with an auxiliary gas inlet,wherein the auxiliary gas inlet can be capable of being supplied withnitrogen gas.

In some exemplary embodiments, the liquid chromatography massspectrometry system can comprise an electrospray ionization sourcehaving an electrospray ionization probe including an electrosprayemitter needle, a sheath gas flow plumbing, and an auxiliary gas flowplumbing.

In some exemplary embodiments, the liquid chromatography massspectrometry system can comprise an electrospray ionization sourcehaving an electrospray ionization probe including an electrosprayemitter needle, a sheath gas flow plumbing, and an auxiliary gas flowplumbing, wherein the electrospray ionization probe can be configured todirect flow in the sheath gas flow plumbing coaxially to theelectrospray emitter needle.

In some exemplary embodiments, the liquid chromatography massspectrometry system can comprise an electrospray ionization sourcehaving an electrospray ionization probe including an electrosprayemitter needle, a sheath gas flow plumbing, and an auxiliary gas flowplumbing, wherein the electrospray ionization probe can be configured todirect flow in the auxiliary gas flow plumbing coaxially to theelectrospray emitter needle.

In some exemplary embodiments, the liquid chromatography massspectrometry system can comprise an electrospray ionization sourcehaving an electrospray ionization probe, wherein the electrosprayionization probe can be a heated electrospray ionization probe.

In some exemplary embodiments, the liquid chromatography massspectrometry system can comprise an electrospray ionization sourcehaving a cap with an inlet line port and a sheath gas inlet line,wherein the sheath gas inlet line can be partially inserted into to theinlet line port.

In some exemplary embodiments, the liquid chromatography massspectrometry system can comprise an electrospray ionization sourcehaving a cap with an outlet line port and a modified desolvation gasoutlet line, wherein the modified desolvation gas outlet line can bepartially inserted into to the outlet line port.

In some exemplary embodiments, the liquid chromatography massspectrometry system can comprise an electrospray ionization sourcehaving a container comprising a cap with an inlet line port and anoutlet line port, a sheath gas inlet line, and a modified desolvationgas outlet line, wherein the electrospray ionization source can beconfigured to allow a flow of a sheath gas from the sheath gas inletline through the container into the desolvation gas outlet line.

In some exemplary embodiments the liquid chromatography massspectrometry system can comprise an electrospray ionization sourcehaving a container comprising a cap with an inlet line port and anoutlet line port, a sheath gas inlet line, and a modified desolvationgas outlet line, wherein the electrospray ionization source can beconfigured to allow a flow of a sheath gas from the sheath gas inletline through the container having an organic solvent into thedesolvation gas outlet line.

In some exemplary embodiments, the liquid chromatography massspectrometry system can comprise an electrospray ionization sourcehaving a container comprising a cap with an inlet line port and anoutlet line port, a sheath gas inlet line, and a modified desolvationgas outlet line, wherein the electrospray ionization source can beconfigured to allow a flow of a sheath gas from the sheath gas inletline through the container having an organic solvent and an additionalcomponent into the desolvation gas outlet line.

In some exemplary embodiments, the liquid chromatography massspectrometry system can comprise electrospray ionization capable ofproviding an electrospray with a solvent flow rate of greater than about5 μL/min.

In some exemplary embodiments, the liquid chromatography massspectrometry system can comprise an electrospray ionization sourcehaving a container surrounded by a second container.

In some exemplary embodiments, the liquid chromatography massspectrometry system can comprise an electrospray ionization sourcehaving a container, wherein the container can be a pressure resistantcontainer.

In some exemplary embodiments, the liquid chromatography massspectrometry system can comprise analyzing the ions using a massspectrometer to identify the components of the protein to characterizethe protein, wherein the mass spectrometer can be a tandem massspectrometer.

This disclosure, at least in part, provides an electrospray ionizationsource, comprising a container having a cap with an inlet line port andan outlet line port, a sheath gas inlet, a modified desolvation gasoutlet line, and an electrospray ionization probe with a sheath gasinlet.

In some exemplary embodiments, the electrospray ionization source cancomprise a container having a cap with an inlet line port and an outletline port and a sheath gas inlet line capable of providing sheath gas tothe inlet line port.

In some exemplary embodiments, the electrospray ionization source cancomprise a container having a cap with an inlet line port and an outletline port and a modified desolvation gas outlet line capable ofconnecting the outlet line port to a sheath gas inlet of an electrosprayionization probe.

In some exemplary embodiments, the electrospray ionization source cancomprise an electrospray ionization probe that can be capable of beingrun in a positive polarity mode.

In some exemplary embodiments, the electrospray ionization source cancomprise an electrospray ionization probe that can be capable of beingrun in a negative polarity mode.

In some exemplary embodiments, the electrospray ionization source cancomprise a container capable of being filled with an organic solvent.

In some exemplary embodiments, the electrospray ionization source cancomprise a container capable of being filled with an organic solvent andan acid.

In some exemplary embodiments, the electrospray ionization source cancomprise a container capable of being filled with an organic solvent anda base.

In some exemplary embodiments, the electrospray ionization source cancomprise an electrospray ionization probe with an auxiliary gas inlet.

In some exemplary embodiments, the electrospray ionization source cancomprise an electrospray ionization probe with an electrospray emitterneedle.

In some exemplary embodiments, the electrospray ionization source cancomprise a container having a cap with an inlet line port and an outletline port and a sheath gas inlet line capable of providing nitrogen gasto the inlet line port.

In some exemplary embodiments, the electrospray ionization source cancomprise an electrospray ionization probe with an auxiliary gas inlet,wherein the auxiliary gas inlet can be capable of being supplied with anauxiliary gas.

In some exemplary embodiments, the electrospray ionization source cancomprise an electrospray ionization probe with an auxiliary gas inlet,wherein the auxiliary gas inlet can be capable of being supplied withnitrogen gas.

In some exemplary embodiments, the electrospray ionization source cancomprise an electrospray ionization probe including an electrosprayemitter needle, a sheath gas flow plumbing, and an auxiliary gas flowplumbing.

In some exemplary embodiments, the electrospray ionization source cancomprise an electrospray ionization probe including an electrosprayemitter needle, a sheath gas flow plumbing, and an auxiliary gas flowplumbing, wherein the electrospray ionization probe can be configured todirect flow in the sheath gas flow plumbing coaxially to theelectrospray emitter needle.

In some exemplary embodiments, the electrospray ionization source cancomprise an electrospray ionization probe including an electrosprayemitter needle, a sheath gas flow plumbing, and an auxiliary gas flowplumbing, wherein the electrospray ionization probe can be configured todirect flow in the auxiliary gas flow plumbing coaxially to theelectrospray emitter needle.

In some exemplary embodiments, the electrospray ionization source cancomprise an electrospray ionization probe, wherein the electrosprayionization probe can be a heated electrospray ionization probe.

In some exemplary embodiments, the electrospray ionization source cancomprise a container having a cap with an inlet line port and a sheathgas inlet line, wherein the sheath gas inlet line can be partiallyinserted into to the inlet line port.

In some exemplary embodiments, the electrospray ionization source cancomprise an electrospray ionization source having a cap with an outletline port and a modified desolvation gas outlet line, wherein themodified desolvation gas outlet line can be partially inserted into tothe outlet line port.

In some exemplary embodiments, the electrospray ionization source cancomprise a container comprising a cap with an inlet line port and anoutlet line port, a sheath gas inlet line, and a modified desolvationgas outlet line, wherein the electrospray ionization source can beconfigured to allow a flow of a sheath gas from the sheath gas inletline through the container into the desolvation gas outlet line.

In some exemplary embodiments, the electrospray ionization source cancomprise a container comprising a cap with an inlet line port and anoutlet line port, a sheath gas inlet line, and a modified desolvationgas outlet line, wherein the electrospray ionization source can beconfigured to allow a flow of a sheath gas from the sheath gas inletline through the container having an organic solvent into thedesolvation gas outlet line.

In some exemplary embodiments, the electrospray ionization source cancomprise a container comprising a cap with an inlet line port and anoutlet line port, a sheath gas inlet line, and a modified desolvationgas outlet line, wherein the electrospray ionization source can beconfigured to allow a flow of a sheath gas from the sheath gas inletline through the container having an organic solvent and an additionalcomponent into the desolvation gas outlet line.

In some exemplary embodiments the electrospray ionization source cancomprise a container surrounded by a second container.

In some exemplary embodiments, the electrospray ionization source cancomprise a container, wherein the container can be a pressure resistantcontainer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic illustration of the difference between top-downand bottom-up proteomics.

FIG. 2 shows a representation of an electrospray ionization source,which can be used for desolvation gas modification on a massspectrometer according to one exemplary embodiment.

FIG. 3 shows base peak chromatograms from the LC-MS analysis of NISTmAbdigests using formic acid (FA)-based method according to one exemplaryembodiment.

FIG. 4 shows base peak chromatograms from the LC-MS analysis of NISTmAbdigests using trifluoroacetic acid-based method according to oneexemplary embodiment.

FIG. 5 shows base peak chromatograms from the LC-MS analysis of NISTmAbdigests using trifluoroacetic acid (TFA)-based method with PA/IPAmodified desolvation gas according to one exemplary embodiment, whereinthe MS signal from TFA control experiments have been amplified two timesfor better visualization

FIG. 6 shows MS intensities of six representative tryptic peptides fromNISTmAb digests using control method (run 1-2) and desolvation gasmodified method (run 3-39) wherein the desolvation gas modified methodis performed according to one exemplary embodiment.

FIG. 7 shows the mass spectra of reduced heavy chain of NISTmAb usingthe control method carried out according to one exemplary embodiment.

FIG. 8 shows the mass spectra of reduced heavy chain of NISTmAb usingthe charge reduction method carried out according to one exemplaryembodiment.

FIG. 9 shows deconvoluted mass spectra of reduced heavy chain of NISTmAbusing control method (top panel) and charge reduction method (bottompanel) carried out according to one exemplary embodiment.

FIG. 10 shows deconvoluted mass spectra of reduced light chain ofNISTmAb using control method (top panel) and charge reduction method(bottom panel) carried out according to one exemplary embodiment.

FIG. 11 shows the raw mass spectra of a highly heterogeneous proteinwith multiple O-glycans using a LC-MS analysis acquired using normalmethod (top panel) and charge reduction method (bottom panel) accordingto one exemplary embodiment.

FIG. 12 shows the deconvoluted mass spectrum of a highly heterogeneousprotein with multiple O-glycans using a LC-MS analysis acquired usingcharge reduction method according to one exemplary embodiment.

DETAILED DESCRIPTION

LC-MS based peptide mapping analysis is routinely applied to confirm theprimary sequence of protein biopharmaceuticals, where a protein moleculeis first hydrolyzed into small peptide fragments using a protease withknown specificity (although non-specific protease can also be appliedoccasionally), and then the amino acid sequence of each peptide fragmentis determined by LC-MS/MS analysis taking into consideration of the cDNApredicted sequence and the specificity of the protease used (Dick et al.Journal of chromatography. B, Analytical technologies in the biomedicaland life sciences 2009, 877, 230-236; Bongers et al. Journal ofpharmaceutical and biomedical analysis 2000, 21, 1099-1128; Mouchahoirand Schiel Analytical and bioanalytical chemistry 2018, 410, 2111-2126).Data from peptide mapping analysis could also be utilized to identifyand quantify post-translational modifications, confirm the disulfidebond linkages and even detect amino acid substitution events present atvery low levels (<0.1%) (Zeck et al. PloS one 2012, 7, e40328.). Duringpeptide mapping analysis of protein biopharmaceuticals, LC-MS is oftenperformed in combination with ultraviolet (UV) detection to generateso-called UV fingerprints, which alone can be used as an identificationassay during quality control (QC) and drug release. To effectivelyseparate peptides on a reversed-phase column with good peak shape,trifluoroacetic acid (TFA) is commonly used as a mobile phase modifierdue to its excellent ion pairing ability (Shibue, et al. Journal ofchromatography. A 2005, 1080, 68-75).

However, TFA is also notoriously known for its ion suppression effectsduring the electrospray (ESI) process due to the increased surfacetension as well as its ability to form ion pairs with analytes in gasphase, thus leading to significantly decreased MS sensitivity (Annesley,T. M. Clinical chemistry 2003, 49, 1041-1044.). Over the past twodecades, different strategies have been investigated and implemented toalleviate the MS sensitivity loss related to TFA. For example, modifyingthe TFA-containing mobile phases with acetic acid or propionic acid hasdemonstrated a significant MS signal enhancement without compromisingthe chromatography integrity for bioanalysis of some basic compounds(Shou and Naidong. Journal of chromatography. B, Analytical technologiesin the biomedical and life sciences 2005, 825, 186-192). Post-columnaddition of a mixture of propionic acid and isopropanol is anothercommonly used strategy which does not require the modification of the LCmethod. However, this setup does require additional pumps, consume largequantity of chemicals and is not suitable for continuous analysis oflarge sample sets (Apffel et al. Journal of chromatography. A 1995, 712,177-190). Acid vapor assisted ESI within an enclosed spray chamber is asolution to counteract the signal suppression effects of TFA (Chen etal. Chemical communications 2015, 51, 14758-14760). However, its utilityin protein biopharmaceutical characterization has been limited so far,presumably due to the requirement of a special ESI source.

Finally, the advances in reversed-phase column chemistry, particularlythe development of charged-surface C18 stationary phases, significantlyreduced the dependence of using TFA to achieve good peak shape duringpeptide mapping analysis (Lauber et al. J. Analytical chemistry 2013,85, 6936-6944). Replacing TFA with a MS-friendly mobile phase modifier(e.g. formic acid), however, will inevitably reduce the retention ofmost peptides, rendering some short and hydrophilic peptidesundetectable due to co-elution with the solvent front, resulting indecreased sequence coverage. Nevertheless, until those new columns havebeen routinely and widely adopted in protein biopharmaceuticalcharacterization, TFA-based LC-MS method is still the mainstream inpeptide mapping analysis. Considering the limitations of existingmethods, an effective and efficient system and method forcharacterization of proteins was developed as disclosed herein. A simpleapproach to counteract TFA ion suppression during LC-MS analysis wascarried out by modifying the desolvation gas with acid/base vapor andisopropanol.

Unless described otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although any methodsand materials similar or equivalent to those described herein can beused in the practice or testing, particular methods and materials arenow described. All publications mentioned are hereby incorporated byreference.

The term “a” should be understood to mean “at least one”; and the terms“about” and “approximately” should be understood to permit standardvariation as would be understood by those of ordinary skill in the art;and where ranges are provided, endpoints are included.

Protein biopharmaceuticals are required to show high levels of potency,purity, and low level of structural heterogeneity. Structuralheterogeneity often affects the bioactivity and efficacy of a drug.Therefore, characterizing and quantifying the protein and/or theimpurities is important in pharmaceutical drug development.

In some exemplary embodiments, the disclosure provides a method forcharacterizing an impurity in a sample.

As used herein, the term “protein” includes any amino acid polymerhaving covalently linked amide bonds. Proteins comprise one or moreamino acid polymer chains, generally known in the art as “polypeptides.”“Polypeptide” refers to a polymer composed of amino acid residues,related naturally occurring structural variants, and syntheticnon-naturally occurring analogs thereof linked via peptide bonds,related naturally occurring structural variants, and syntheticnon-naturally occurring analogs thereof. “Synthetic peptides orpolypeptides’ refers to a non-naturally occurring peptide orpolypeptide. Synthetic peptides or polypeptides can be synthesized, forexample, using an automated polypeptide synthesizer. Various solid phasepeptide synthesis methods are known to those of skill in the art. Aprotein may contain one or multiple polypeptides to form a singlefunctioning biomolecule. A protein can include any of bio-therapeuticproteins, recombinant proteins used in research or therapy, trapproteins and other chimeric receptor Fc-fusion proteins, chimericproteins, antibodies, monoclonal antibodies, polyclonal antibodies,human antibodies, and bispecific antibodies. In another exemplaryaspect, a protein can include antibody fragments, nanobodies,recombinant antibody chimeras, cytokines, chemokines, peptide hormones,and the like. Proteins may be produced using recombinant cell-basedproduction systems, such as the insect bacculovirus system, yeastsystems (e.g., Pichia sp.), mammalian systems (e.g., CHO cells and CHOderivatives like CHO-K1 cells). For a recent review discussingbiotherapeutic proteins and their production, see Ghaderi et al.,“Production platforms for biotherapeutic glycoproteins. Occurrence,impact, and challenges of non-human sialylation,” (Biotechnol. Genet.Eng. Rev. (2012) 147-75). In some embodiments, proteins comprisemodifications, adducts, and other covalently linked moieties. Thosemodifications, adducts and moieties include for example avidin,streptavidin, biotin, glycans (e.g., N-acetylgalactosamine, galactose,neuraminic acid, N-acetylglucosamine, fucose, mannose, and othermonosaccharides), PEG, polyhistidine, FLAGtag, maltose binding protein(MBP), chitin binding protein (CBP), glutathione-S-transferase (GST)myc-epitope, fluorescent labels and other dyes, and the like. Proteinscan be classified on the basis of compositions and solubility and canthus include simple proteins, such as, globular proteins and fibrousproteins; conjugated proteins, such as, nucleoproteins, glycoproteins,mucoproteins, chromoproteins, phosphoproteins, metalloproteins, andlipoproteins; and derived proteins, such as, primary derived proteinsand secondary derived proteins.

In some exemplary embodiments, the protein can be an antibody, abispecific antibody, a multispecific antibody, antibody fragment,monoclonal antibody, or combinations thereof.

The term “antibody,” as used herein includes immunoglobulin moleculescomprising four polypeptide chains, two heavy (H) chains and two light(L) chains inter-connected by disulfide bonds, as well as multimersthereof (e.g., IgM). Each heavy chain comprises a heavy chain variableregion (abbreviated herein as HCVR or V_(H)) and a heavy chain constantregion. The heavy chain constant region comprises three domains, C_(H)1,C_(H)2 and C_(H)3. Each light chain comprises a light chain variableregion (abbreviated herein as LCVR or V_(L)) and a light chain constantregion. The light chain constant region comprises one domain (C_(L)1).The V_(H) and V_(L) regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDRs),interspersed with regions that are more conserved, termed frameworkregions (FR). Each V_(H) and V_(L) is composed of three CDRs and fourFRs, arranged from amino-terminus to carboxy-terminus in the followingorder: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments ofthe invention, the FRs of the anti-big-ET-1 antibody (or antigen-bindingportion thereof) may be identical to the human germline sequences, ormay be naturally or artificially modified. An amino acid consensussequence may be defined based on a side-by-side analysis of two or moreCDRs. The term “antibody,” as used herein, also includes antigen-bindingfragments of full antibody molecules. The terms “antigen-bindingportion” of an antibody, “antigen-binding fragment” of an antibody, andthe like, as used herein, include any naturally occurring, enzymaticallyobtainable, synthetic, or genetically engineered polypeptide orglycoprotein that specifically binds an antigen to form a complex.Antigen-binding fragments of an antibody may be derived, e.g., from fullantibody molecules using any suitable standard techniques such asproteolytic digestion or recombinant genetic engineering techniquesinvolving the manipulation and expression of DNA encoding antibodyvariable and optionally constant domains. Such DNA is known and/or isreadily available from, e.g., commercial sources, DNA libraries(including, e.g., phage-antibody libraries), or can be synthesized. TheDNA may be sequenced and manipulated chemically or by using molecularbiology techniques, for example, to arrange one or more variable and/orconstant domains into a suitable configuration, or to introduce codons,create cysteine residues, modify, add or delete amino acids, etc.

As used herein, an “antibody fragment” includes a portion of an intactantibody, such as, for example, the antigen-binding or variable regionof an antibody. Examples of antibody fragments include, but are notlimited to, a Fab fragment, a Fab′ fragment, a F(ab′)2 fragment, a scFvfragment, a Fv fragment, a dsFv diabody, a dAb fragment, a Fd′ fragment,a Fd fragment, and an isolated complementarity determining region (CDR)region, as well as triabodies, tetrabodies, linear antibodies,single-chain antibody molecules, and multi specific antibodies formedfrom antibody fragments. Fv fragments are the combination of thevariable regions of the immunoglobulin heavy and light chains, and ScFvproteins are recombinant single chain polypeptide molecules in whichimmunoglobulin light and heavy chain variable regions are connected by apeptide linker. In some exemplary embodiments, an antibody fragmentcontains sufficient amino acid sequence of the parent antibody of whichit is a fragment that it binds to the same antigen as does the parentantibody; in some exemplary embodiments, a fragment binds to the antigenwith a comparable affinity to that of the parent antibody and/orcompetes with the parent antibody for binding to the antigen. Anantibody fragment may be produced by any means. For example, an antibodyfragment may be enzymatically or chemically produced by fragmentation ofan intact antibody and/or it may be recombinantly produced from a geneencoding the partial antibody sequence. Alternatively or additionally,an antibody fragment may be wholly or partially synthetically produced.An antibody fragment may optionally comprise a single chain antibodyfragment. Alternatively or additionally, an antibody fragment maycomprise multiple chains that are linked together, for example, bydisulfide linkages. An antibody fragment may optionally comprise amulti-molecular complex. A functional antibody fragment typicallycomprises at least about 50 amino acids and more typically comprises atleast about 200 amino acids.

The phrase “bispecific antibody” includes an antibody capable ofselectively binding two or more epitopes. Bispecific antibodiesgenerally comprise two different heavy chains, with each heavy chainspecifically binding a different epitope—either on two differentmolecules (e.g., antigens) or on the same molecule (e.g., on the sameantigen). If a bispecific antibody is capable of selectively binding twodifferent epitopes (a first epitope and a second epitope), the affinityof the first heavy chain for the first epitope will generally be atleast one to two or three or four orders of magnitude lower than theaffinity of the first heavy chain for the second epitope, and viceversa. The epitopes recognized by the bispecific antibody can be on thesame or a different target (e.g., on the same or a different protein).Bispecific antibodies can be made, for example, by combining heavychains that recognize different epitopes of the same antigen. Forexample, nucleic acid sequences encoding heavy chain variable sequencesthat recognize different epitopes of the same antigen can be fused tonucleic acid sequences encoding different heavy chain constant regions,and such sequences can be expressed in a cell that expresses animmunoglobulin light chain. A typical bispecific antibody has two heavychains each having three heavy chain CDRs, followed by a C_(H)1 domain,a hinge, a C_(H)2 domain, and a C_(H)3 domain, and an immunoglobulinlight chain that either does not confer antigen-binding specificity butthat can associate with each heavy chain, or that can associate witheach heavy chain and that can bind one or more of the epitopes bound bythe heavy chain antigen-binding regions, or that can associate with eachheavy chain and enable binding or one or both of the heavy chains to oneor both epitopes. BsAbs can be divided into two major classes, thosebearing an Fc region (IgG-like) and those lacking an Fc region, thelatter normally being smaller than the IgG and IgG-like bispecificmolecules comprising an Fc. The IgG-like bsAbs can have differentformats, such as, but not limited to triomab, knobs into holes IgG (kihIgG), crossMab, orth-Fab IgG, Dual-variable domains Ig (DVD-Ig),Two-in-one or dual action Fab (DAF), IgG-single-chain Fv (IgG-scFv), orκλ-bodies. The non-IgG-like different formats include Tandem scFvs,Diabody format, Single-chain diabody, tandem diabodies (TandAbs),Dual-affinity retargeting molecule (DART), DART-Fc, nanobodies, orantibodies produced by the dock-and-lock (DNL) method. Fan et al. andKontermann and Brinkmann present a detailed review on bispecificantibody (Fan et al. “Bispecific antibodies and their applications” J.Hematol. Oncol. (2015) 8:130; Kontermann and Brinkmann. “Bispecificantibodies” Drug Discov. Today (2015) 20: 838-847). The methods ofproducing BsAbs are not limited to quadroma technology based on thesomatic fusion of two different hybridoma cell lines, chemicalconjugation, which involves chemical cross-linkers, and geneticapproaches utilizing recombinant DNA technology. Examples of bsAbsinclude those disclosed in the following patent applications, which arehereby incorporated by reference in their entirety: U.S. Pat. No.8,586,713, filed Jun. 25, 2010; U.S. Pat. Publication No. 2013/0045492,filed Jun. 5, 2012; U.S. Pat. No. 9,657,102, filed Sep. 19, 2013; U.S.Pat. Publication No. 2016/0024147, filed Jul. 24, 2015; U.S. Pat.Publication No. 2018/0112001, filed Sep. 22, 2017; U.S. Pat. PublicationNo. 2018/0104357, field Sep. 22, 2017; U.S. Pat. Publication No.2017/0174779, filed Dec. 21, 2016; U.S. Pat. Publication No.2017/0174781, filed Dec. 21, 2016; U.S. Pat. No. 10,179,819, filed Jul.29, 2016; and U.S. Pat. Publication No. 2018/0134794, filed Nov. 15,2017. Low levels of homodimer impurities can be present at several stepsduring the manufacturing of bispecific antibodies. The detection of suchhomodimer impurities can be challenging when performed using intact massanalysis due to low abundances of the homodimer impurities and theco-elution of these impurities with main species when carried out usinga regular liquid chromatographic method.

As used herein “multispecific antibody” or “Mab” refers to an antibodywith binding specificities for at least two different antigens. Whilesuch molecules normally will only bind two antigens (i.e. bispecificantibodies, BsAbs), antibodies with additional specificities such astrispecific antibody and KIH Trispecific can also be addressed by thesystem and method disclosed herein.

The term “monoclonal antibody” as used herein is not limited toantibodies produced through hybridoma technology. A monoclonal antibodycan be derived from a single clone, including any eukaryotic,prokaryotic, or phage clone, by any means available or known in the art.Monoclonal antibodies useful with the present disclosure can be preparedusing a wide variety of techniques known in the art including the use ofhybridoma, recombinant, and phage display technologies, or a combinationthereof.

During many stages of production of biopharmaceuticals, impurities canbe formed. Biotechnology-derived impurities can be very difficult tocharacterize and quantify, because they often are present at very lowlevels, and because they can represent very complicated species ormixtures of species. It can also be very difficult to obtain anauthentic reference standard of the impurity peaks. However, to fullycharacterize a trace amount of an impurity protein becomes a timeconsuming, lengthy, and often very expensive process. Often, theimpurity can include variants, isoforms, degradation products,product-related impurities, process-related, minor post translationalmodifications, aggregates, or clipped fragments of the intactrecombinant protein.

In some exemplary embodiments, the disclosure provides a method forcharacterizing a protein in a sample.

As used herein, the term “impurity” can include any undesirable proteinpresent in the biopharmaceutical product. Impurity can include processand product-related impurities. The impurity can further be of knownstructure, partially characterized, or unidentified. Process-relatedimpurities can be derived from the manufacturing process and can includethe three major categories: cell substrate-derived, cell culture-derivedand downstream derived. Cell substrate-derived impurities include, butare not limited to, proteins derived from the host organism and nucleicacid (host cell genomic, vector, or total DNA). Cell culture-derivedimpurities include, but are not limited to, inducers, antibiotics,serum, and other media components. Downstream-derived impuritiesinclude, but are not limited to, enzymes, chemical and biochemicalprocessing reagents (e.g., cyanogen bromide, guanidine, oxidizing andreducing agents), inorganic salts (e.g., heavy metals, arsenic,nonmetallic ion), solvents, carriers, ligands (e.g., monoclonalantibodies), and other leachables. Product-related impurities (e.g.,precursors, certain degradation products) can be molecular variantsarising during manufacture and/or storage that do not have propertiescomparable to those of the desired product with respect to activity,efficacy, and safety. Such variants may need considerable effort inisolation and characterization in order to identify the type ofmodification(s). Product-related impurities can include truncated forms,modified forms, and aggregates. Truncated forms are formed by hydrolyticenzymes or chemicals which catalyze the cleavage of peptide bonds.Modified forms include, but are not limited to, deamidated, isomerized,mismatched S-S linked, oxidized, or altered conjugated forms (e.g.,glycosylation, phosphorylation). Modified forms can also include anypost-translational modification form. Aggregates include dimers andhigher multiples of the desired product. (Q6B Specifications: TestProcedures and Acceptance Criteria for Biotechnological/BiologicalProducts, ICH August 1999, U.S. Dept. of Health and Humans Services).

As used herein, the general term “post-translational modifications” or“PTMs” refer to covalent modifications that polypeptides undergo, eitherduring (co-translational modification) or after (post-translationalmodification) their ribosomal synthesis. PTMs are generally introducedby specific enzymes or enzyme pathways. Many occur at the site of aspecific characteristic protein sequence (e.g., signature sequence)within the protein backbone. Several hundred PTMs have been recorded,and these modifications invariably influence some aspect of a protein'sstructure or function (Walsh, G. “Proteins” (2014) second edition,published by Wiley and Sons, Ltd., ISBN: 9780470669853). The variouspost-translational modifications include, but are not limited to,cleavage, N-terminal extensions, protein degradation, acylation of theN-terminus, biotinylation (acylation of lysine residues with a biotin),amidation of the C-terminal, glycosylation, iodination, covalentattachment of prosthetic groups, acetylation (the addition of an acetylgroup, usually at the N-terminus of the protein), alkylation (theaddition of an alkyl group (e.g. methyl, ethyl, propyl) usually atlysine or arginine residues), methylation, adenylation,ADP-ribosylation, covalent cross links within, or between, polypeptidechains, sulfonation, prenylation, Vitamin C dependent modifications(proline and lysine hydroxylations and carboxy terminal amidation),Vitamin K dependent modification wherein Vitamin K is a cofactor in thecarboxylation of glutamic acid residues resulting in the formation of aγ-carboxyglutamate (a glu residue), glutamylation (covalent linkage ofglutamic acid residues), glycylation (covalent linkage glycineresidues), glycosylation (addition of a glycosyl group to eitherasparagine, hydroxylysine, serine, or threonine, resulting in aglycoprotein), isoprenylation (addition of an isoprenoid group such asfarnesol and geranylgeraniol), lipoylation (attachment of a lipoatefunctionality), phosphopantetheinylation (addition of a4′-phosphopantetheinyl moiety from coenzyme A, as in fatty acid,polyketide, non-ribosomal peptide and leucine biosynthesis),phosphorylation (addition of a phosphate group, usually to serine,tyrosine, threonine or histidine), and sulfation (addition of a sulfategroup, usually to a tyrosine residue). The post-translationalmodifications that change the chemical nature of amino acids include,but are not limited to, citrullination (e.g., the conversion of arginineto citrulline by deimination), and deamidation (e.g., the conversion ofglutamine to glutamic acid or asparagine to aspartic acid). Thepost-translational modifications that involve structural changesinclude, but are not limited to, formation of disulfide bridges(covalent linkage of two cysteine amino acids) and proteolytic cleavage(cleavage of a protein at a peptide bond). Certain post-translationalmodifications involve the addition of other proteins or peptides, suchas ISGylation (covalent linkage to the ISG15 protein(Interferon-Stimulated Gene)), SUMOylation (covalent linkage to the SUMOprotein (Small Ubiquitin-related MOdifier)) and ubiquitination (covalentlinkage to the protein ubiquitin). Seehttp://www.uniprot.org/docs/ptmlist for a more detailed controlledvocabulary of PTMs curated by UniProt.

“Variant protein” or “protein variant”, or “variant” as used herein caninclude a protein that differs from a target protein by virtue of atleast one amino acid modification. Protein variant may refer to theprotein itself, a composition comprising the protein, or the aminosequence that encodes it. Preferably, the protein variant has at leastone amino acid modification compared to the parent protein, e.g. fromabout one to about ten amino acid modifications, and preferably fromabout one to about five amino acid modifications compared to the parent.The protein variant sequence herein will preferably possess at leastabout 80% homology with a parent protein sequence, and most preferablyat least about 90% homology, more preferably at least about 95%homology.

Comprehensive characterization of proteins can satisfy safety standardsset by regulatory agencies and help to ensure protein drug efficacy.Proteomics approaches are thus important in the biopharmaceuticalindustry where they aid in the identity confirmation of a protein,monitoring impurities, monitoring protein modifications such as PTMs andprotein variants, monitoring degradative events such as oxidation ordeamidation, etc. Proteomics approaches can be discriminated by thelevel at which analysis takes place (See FIG. 1).

“Intact mass analysis” as used herein includes top-down experimentswherein a protein is characterized as an intact protein (See FIG. 1,left panel). Intact mass analysis can reduce sample preparation to aminimum and preserve information that can sometimes get lost in otherproteomics strategies, such as the connectivity of multiple PTMs.

Some proteomics experiments rely on digestion of the protein intopeptides prior to MS analysis. “Peptide mapping analysis” as used hereinincludes experiments wherein the protein undergoes digestion followed byseparation of the resulting peptides and their analysis, preferablyusing LC-MS (See FIG. 1, right panel). In some exemplary embodiments,peptide mapping analysis can be applied to confirm the primary sequenceof protein biopharmaceuticals, where a protein molecule can be firsthydrolyzed into small peptide fragments using a hydrolyzing agent andthen the amino acid sequence of each peptide fragment is determined byLC-MS/MS analysis taking into consideration of the cDNA predictedsequence and the specificity of the protease used. Data from peptidemapping analysis could also be utilized to identify and quantifypost-translational modifications, confirm the disulfide bond linkagesand even detect amino acid substitution events present at very lowlevels (<0.1%) (Zeck et al. PloS one 2012, 7, e40328). During peptidemapping analysis of protein biopharmaceuticals, LC-MS can be oftenperformed in combination with ultraviolet (UV) detection to generateso-called UV fingerprints, which alone can be used as an identificationassay during quality control (QC) and drug release.

As used herein, the term “digestion” refers to hydrolysis of one or morepeptide bonds of a protein. There are several approaches to carrying outdigestion of a protein in a sample using an appropriate hydrolyzingagent, for example, enzymatic digestion or non-enzymatic digestion. Asused herein, the term “hydrolyzing agent” refers to any one orcombination of a large number of different agents that can performdigestion of a protein. Non-limiting examples of hydrolyzing agents thatcan carry out enzymatic digestion include trypsin, endoproteinase Arg-C,endoproteinase Asp-N, endoproteinase Glu-C, outer membrane protease T(OmpT), immunoglobulin-degrading enzyme of Streptococcus pyogenes(IdeS), chymotrypsin, pepsin, thermolysin, papain, pronase, and proteasefrom Aspergillus Saitoi. Non-limiting examples of hydrolyzing agentsthat can carry out non-enzymatic digestion include the use of hightemperature, microwave, ultrasound, high pressure, infrared, solvents(non-limiting examples are ethanol and acetonitrile), immobilized enzymedigestion (IMER), magnetic particle immobilized enzymes, and on-chipimmobilized enzymes. For a recent review discussing the availabletechniques for protein digestion see Switazar et al., “ProteinDigestion: An Overview of the Available Techniques and RecentDevelopments” (J. Proteome Research 2013, 12, 1067-1077). One or acombination of hydrolyzing agents can cleave peptide bonds in a proteinor polypeptide, in a sequence-specific manner, generating a predictablecollection of shorter peptides.

Several approaches are available that can be used to digest a protein.One of the widely accepted methods for digestion of proteins in a sampleinvolves the use of proteases. Many proteases are available and each ofthem has their own characteristics in terms of specificity, efficiency,and optimum digestion conditions. Proteases refer to both endopeptidasesand exopeptidases, as classified based on the ability of the protease tocleave at non-terminal or terminal amino acids within a peptide.Alternatively, proteases also refer to the six distinctclasses—aspartic, glutamic, and metalloproteases, cysteine, serine, andthreonine proteases, as classified on the mechanism of catalysis. Theterms “protease” and “peptidase” are used interchangeably to refer toenzymes which hydrolyze peptide bonds. Proteases can also be classifiedinto specific and non-specific proteases. As used herein, the term“specific protease” refers to a protease with an ability to cleave thepeptide substrate at a specific amino acid side chain of a peptide. Asused herein, the term “non-specific protease” refers to a protease witha reduced ability to cleave the peptide substrate at a specific aminoacid side chain of a peptide. A cleavage preference may be determinedbased on the ratio of the number of a particular amino acid as the siteof cleavage to the total number of cleaved amino acids in the proteinsequences

The protein can optionally be prepared before characterizing. In someexemplary embodiments, the protein preparation includes a step ofprotein digestion. In some specific exemplary embodiments, the proteinpreparation includes a step of protein digestion, wherein the proteindigestion can be carried out using trypsin.

In some exemplary embodiments, the protein preparation can include astep for denaturing the protein, reducing the protein, buffering theprotein, and/or desalting the sample, before the step of proteindigestion. These steps can be accomplished in any suitable manner asdesired.

To provide characterization of different protein attributes using eitherpeptide mapping analysis or intact mass analysis, a wide variety ofLC-MS based assays can be performed.

As used herein, the term “liquid chromatography” refers to a process inwhich a chemical mixture carried by a liquid can be separated intocomponents as a result of differential distribution of the chemicalentities as they flow around or over a stationary liquid or solid phase.Non-limiting examples of liquid chromatography include reverse phaseliquid chromatography, ion-exchange chromatography, size exclusionchromatography, affinity chromatography, and hydrophobic chromatography.

As used herein, the term “mass spectrometer” refers to a device capableof detecting specific molecular species and accurately measuring theirmasses. The term can be meant to include any molecular detector intowhich a polypeptide or peptide may be eluted for detection and/orcharacterization. A mass spectrometer consists of three major parts: theion source, the mass analyzer, and the detector. The role of the ionsource is to create gas phase ions. Analyte atoms, molecules, orclusters can be transferred into gas phase and ionized eitherconcurrently (as in electrospray ionization). The choice of ion sourcedepends on the application.

As used herein, the term “electrospray ionization” or “ESI” refers tothe process of spray ionization in which either cations or anions insolution are transferred to the gas phase via formation and desolvationat atmospheric pressure of a stream of highly charged droplets thatresult from applying a potential difference between the tip of theelectrospray emitter needle containing the solution and a counterelectrode. There are three major steps in the production of gas-phaseions from electrolyte ions in solution. These are: (a) production ofcharged droplets at the ES infusion tip; (b) shrinkage of chargeddroplets by solvent evaporation and repeated droplet disintegrationsleading to small highly charged droplets capable of producing gas-phaseions; and (c) the mechanism by which gas-phase ions are produced fromvery small and highly charged droplets. Stages (a)-(c) generally occurin the atmospheric pressure region of the apparatus.

As used herein, the term “electrospray ionization source” refers to anelectrospray ionization system that can be compatible with a massspectrometer used for mass analysis of protein. In electrosprayionization, an electrospray emitter needle has its orifice positionedclose to the entrance orifice of a spectrometer. A sample, containingthe protein of interest, can be pumped through the electrospray emitterneedle. An electric potential between the electrospray emitter needleorifice and an orifice leading to the mass analyzer forms a spray(“electrospray”) of the solution. The electrospray can be carried out atatmospheric pressure and provides highly charged droplets of thesolution. The electrospray ionization source can be configured tooperate in any of several atmospheric pressure ionization (API) modes,including electrospray ionization (ESI), heated-electrospray ionization(H-ESI), and atmospheric pressure chemical ionization (APCI), andatmospheric pressure photo-ionization (APPI). The electrospray infusionsetup can optionally be automated to carry out sample aspiration, sampledispensing, sample delivery, and/or for spraying the sample. Theelectrospray ionization probe can produce charged aerosol droplets thatcontain sample ions. The ESI probe can accommodate liquid flows of 5μL/min to 1 mL/min without splitting.

The ESI probe can include the ESI sample tube, the electrospray emitterneedle, a nozzle, and a manifold. Sample and solvent can enter the ESIprobe through the sample tube. The sample tube can be a short section ofOD fused-silica tubing that extends from the stainless steel groundingto the end of the ESI needle. The electrospray emitter needle, to whicha large negative or positive voltage can be applied, can spray thesample solution into a fine mist of charged droplets. The ESI nozzle candirect the flow of sheath gas and auxiliary gas at the droplets. The ESImanifold can house an ESI nozzle, an electrospray emitter needle, asheath gas inlet, an auxiliary gas inlet, a sheath gas plumbing, and anauxiliary gas plumbing. The sheath gas plumbing and auxiliary gasplumbing can deliver dry gas to the nozzle. The sheath gas inlet andauxiliary gas inlet in the manifold can be connected to sheath gas inletline and auxiliary gas inlet line, respectively. Alternatively, thesheath gas inlet and auxiliary gas inlet in the manifold can beconnected to modified desolvation gas outlet line and auxiliary gasinlet line, respectively. The connection of gas inlets with the gasinlet lines can be performed using adapter fittings.

The term “nanoelectrospray” or “nanospray” as used herein refers toelectrospray ionization at a very low solvent flow rate, typicallyhundreds of nanoliters per minute of sample solution or lower, oftenwithout the use of an external solvent delivery.

As used herein, “mass analyzer” refers to a device that can separatespecies, that is, atoms, molecules, or clusters, according to theirmass. Non-limiting examples of mass analyzers that could be employed forfast protein sequencing are time-of-flight (TOF), magnetic/electricsector, quadrupole mass filter (Q), quadrupole ion trap (QIT), orbitrap,Fourier transform ion cyclotron resonance (FTICR), and also thetechnique of accelerator mass spectrometry (AMS).

As used herein, “mass-to-charge ratio” or “m/z” is used to denote thedimensionless quantity formed by dividing the mass of an ion in unifiedatomic mass units by its charge number (regardless of sign). In general,the charge state depends on: the method of ionization (as electrosprayionization, ESI tends to promote multiple ionization, which is not asfrequent in MALDI), peptide length (as longer peptides have more groupswhere additional protons can be attached (basic residues)), peptidesequence (as some amino acids (e.g., Arg or Lys) are more susceptible toionization than others), the instrument settings, solvent pH, andsolvent composition.

As used herein, the term “tandem mass spectrometry” refers to atechnique where structural information on sample molecules can beobtained by using multiple stages of mass selection and mass separation.A prerequisite is that the sample molecules can be transferred into gasphase and ionized intact and that they can be induced to fall apart insome predictable and controllable fashion after the first mass selectionstep. Multistage MS/MS, or MS^(n), can be performed by first selectingand isolating a precursor ion (MS²), fragmenting it, isolating a primaryfragment ion (MS³), fragmenting it, isolating a secondary fragment(MS⁴), and so on as long as one can obtain meaningful information or thefragment ion signal is detectable. Tandem MS have been successfullyperformed with a wide variety of analyzer combinations. What analyzersto combine for a certain application can be determined by many differentfactors, such as sensitivity, selectivity, and speed, but also size,cost, and availability. The two major categories of tandem MS methodsare tandem-in-space and tandem-in-time, but there are also hybrids wheretandem-in-time analyzers are coupled in space or with tandem-in-spaceanalyzers.

A tandem-in-space mass spectrometer comprise of an ion source, aprecursor ion activation device, and at least two non-trapping massanalyzers. Specific m/z separation functions can be designed so that inone section of the instrument ions are selected, dissociated in anintermediate region, and the product ions are then transmitted toanother analyzer for m/z separation and data acquisition.

In tandem-in-time mass spectrometer ions produced in the ion source canbe trapped, isolated, fragmented, and m/z separated in the same physicaldevice.

As used herein, the term “quadrupole-Orbitrap hybrid mass spectrometer”refers to a hybrid system made by coupling a quadrupole massspectrometer to an orbitrap mass analyzer. A tandem in-time experimentusing the quadrupole-Orbitrap hybrid mass spectrometer begins withejection of all ions except those within a selected, narrow m/z rangefrom the quadrupole mass spectrometer. The selected ions can be insertedinto orbitrap and fragmented most often by low-energy CID. Fragmentswithin the m/z acceptance range of the trap should remain in the trap,and an MS-MS spectrum can be obtained. Similar hybrid systems can beused for fast protein sequencing, such as, but not limited to QIT-FTICRand Qq-FTICR.

It is understood that the present invention is not limited to any of theaforesaid liquid chromatography, mass spectrometer, and that any liquidchromatography or mass spectrometer can be selected by any suitablemeans.

Characterization of the Protein

The peptides identified by the mass spectrometer can be used assurrogate representatives of the intact protein and their posttranslational modifications. They can be used for proteincharacterization by correlating experimental and theoretical MS/MS data,the latter generated from possible peptides in a protein sequencedatabase. The characterization includes, but is not limited, tosequencing amino acids of the protein fragments, determining proteinsequencing, determining protein de novo sequencing, locatingpost-translational modifications, or identifying post translationalmodifications, or comparability analysis, or combinations thereof.

As used herein, the term “protein de novo sequencing” refers to aprocedure for determination of the amino acid sequence of a peptidewithout relying on the information gained from other sources. Due to thehigh level of sensitivity of mass spectrometry, this technique canprovide vital information that is often beyond the capabilities ofconventional sequencing methods.

As used herein, the term “protein sequence coverage” refers to thepercentage of the protein sequence covered by identified peptides. Thepercent coverage can be calculated by dividing the number of amino acidsin all found peptides by the total number of amino acids in the entireprotein sequence.

As used herein, the term “database” refers to bioinformatic tools whichprovide the possibility of searching the uninterpreted MS-MS spectraagainst all possible sequences in the database(s). Non-limiting examplesof such tools are Mascot (http://www.matrixscience.com), Spectrum Mill(http://www.chem.agilent.com), PLGS (http://www.waters.com), PEAKS(http://www.bioinformaticssolutions.com), Proteinpilot(http://download.appliedbiosystems.com//proteinpilot), Phenyx(http://www.phenyx-ms.com), Sorcerer (http://www.sagenresearch.com),OMSSA (http://www.pubchem.ncbi.nlm.nih.gov/omssa/), X!Tandem(http://www.thegpm.org/TANDEM/), Protein Prospector(http://www.http://prospector.ucsf.edu/prospector/mshome.htm), Byonic(https://www.proteinmetrics.com/products/byonic) or Sequest(http://fields.seripps.edu/sequest).

Exemplary Embodiments

Embodiments disclosed herein provide compositions, methods, and systemsfor the rapid characterization of proteins in a sample.

As used herein, the terms “include,” “includes,” and “including,” aremeant to be non-limiting and are understood to mean “comprise,”“comprises,” and “comprising,” respectively.

In some exemplary embodiments, the disclosure provides a liquidchromatography mass spectrometry system, comprising (i) liquidchromatography device, (ii) an electrospray ionization source and (iii)a mass spectrometry device.

In some exemplary embodiments, this disclosure provides an electrosprayionization source 100, comprising (i) a container 110, (ii) a sheath gasinlet line 120, (iii) a modified desolvation gas outlet line 130, and(iv) an electrospray ionization probe 140 (See FIG. 2).

In some exemplary embodiments, the disclosure provides a method ofcharacterizing a protein in a sample, comprising (i) supplying thesample to an inlet of an electrospray ionization source 100, (ii)generating ions of components of the protein in the sample at an outletof the electrospray ionization source, and (iii) analyzing the ionsusing a mass spectrometer to identify the components of the protein tocharacterize the protein.

Non-limiting examples of the liquid chromatography device can includereverse phase liquid chromatography, ion-exchange chromatography, sizeexclusion chromatography, affinity chromatography,hydrophilic-interaction chromatography, and hydrophobic chromatography.

In some exemplary embodiments, the sheath gas inlet line 120 can be aTeflon tube.

In some exemplary embodiments, the sheath gas inlet line 120 can beflexible stainless steel tubing.

In some exemplary embodiments, the sheath gas inlet line 120 can be atube made using poly ether ether ketone.

In some exemplary embodiments, the modified desolvation gas outlet line130 can be a Teflon tube.

In some exemplary embodiments, the modified desolvation gas outlet line130 can be flexible stainless steel tubing.

In some exemplary embodiments, the modified desolvation gas outlet line130 can be a tube made using poly ether ether ketone.

In some exemplary embodiments, the container 110 can comprise a cap 150.The cap can be safe to sue with mobile phase.

In some exemplary embodiments, the container 110 can comprise a cap 150,wherein the cap can be capable of forming an air-tight seal between thecap and the container. Non-limiting example of caps that can be usedinclude Analytical Sales' Canary-Safe Cap, Restek's Eco-cap bottle top,Restek's Opti-cap bottle top, and VWR's inert mobile phase bottle cap.

In some exemplary embodiments, the cap 150 can comprise of a screw cap.

In some exemplary embodiments, the cap 150 can be made ofPolytetrafluoroethylene (PTFE) material.

In some exemplary embodiments, the cap 150 can further comprise of anO-ring to ensure air-tight seal between cap and the container 110.

In some exemplary embodiments, the cap 150 in the container 110 can haveat least one port for tubing. In one aspect, the cap 150 in thecontainer 110 can have two ports for tubing.

In some exemplary embodiments, the cap 150 in the container 110 can haveat least one inlet port 160.

In some exemplary embodiments, the cap 150 in the container 110 can haveat least one outlet port 170.

In some exemplary embodiments, the electrospray ionization probe 140 ofthe electrospray ionization source 100 can comprise a sheath gas inlet180.

In some exemplary embodiments, the electrospray ionization source 100can comprise a container 110 having a cap 150 with an inlet line port160 and an outlet line port 170 and a sheath gas inlet line 120 capableof providing sheath gas to the inlet line port 160.

In some exemplary embodiments, the electrospray ionization source 100can comprise a container 110 having a cap 150 with an inlet line port160 and an outlet line port 170 and a modified desolvation gas outletline 130 capable of connecting the outlet line port 170 to a sheath gasinlet 180 of an electrospray ionization probe 140.

In some exemplary embodiments, the electrospray ionization probe 140 canbe capable of being run in a positive polarity mode.

In some exemplary embodiments, the electrospray ionization probe 140 canbe capable of being run in a negative polarity mode.

In some exemplary embodiments, the electrospray ionization probe 140with an auxiliary gas inlet 190.

In some exemplary embodiments, the electrospray ionization probe 140 canfurther comprise an electrospray emitter needle.

In some exemplary embodiments, a sheath gas can be provided by a sheathgas source 200 to a container 110 having a cap 150 with an inlet lineport 160 through a sheath gas inlet line 120. Non-limiting examples ofsheath gas include air, nitrogen.

In some exemplary embodiments, an auxiliary gas can be provided by anauxiliary gas source 210 to an auxiliary gas inlet 190 of anelectrospray ionization probe 140 through an auxiliary gas inlet line220. Non-limiting examples of auxiliary gas include air, nitrogen.

In some exemplary embodiments, the sheath gas that can be provided by asheath gas source 200 to a container 110 having a cap 150 with an inletline port 160 through a sheath gas inlet line 120 can be nitrogen gas.

In some exemplary embodiments, the auxiliary gas that can be provided byan auxiliary gas source 210 to an auxiliary gas inlet 190 of anelectrospray ionization probe 140 through an auxiliary gas inlet line220 can be nitrogen gas.

In some exemplary embodiments, the electrospray ionization probe 140 caninclude an electrospray emitter needle, a sheath gas flow plumbing, andan auxiliary gas flow plumbing.

In some exemplary embodiments, the electrospray ionization source 110can comprise an electrospray ionization probe 140 which can include anelectrospray emitter needle, a sheath gas flow plumbing, and anauxiliary gas flow plumbing, wherein the electrospray ionization probecan be configured to direct flow in the sheath gas flow plumbingcoaxially to the electrospray emitter needle.

In some exemplary embodiments, the electrospray ionization probe 140 canbe configured to direct flow in the auxiliary gas flow plumbingcoaxially to the electrospray emitter needle.

In some exemplary embodiments, the electrospray ionization probe 140 canbe a heated electrospray ionization probe.

In some exemplary embodiments, the electrospray ionization probe 140 canbe automated to carry out sample aspiration, sample dispensing, sampledelivery and/or for spraying the sample.

In some exemplary embodiments, the container 110 having a cap 150 withan inlet line port 160 and a sheath gas inlet line 120, wherein thesheath gas inlet line 120 can be partially inserted into to the inletline port 160.

In some exemplary embodiments, the cap 150 with an outlet line port 170and a modified desolvation gas outlet line 130, wherein the modifieddesolvation gas outlet line 130 can be partially inserted into to theoutlet line port 170.

In some exemplary embodiments, the electrospray ionization source 100can be configured to allow a flow of a sheath gas from the sheath gasinlet line 120 through the container 100 into the desolvation gas outletline 130.

In some exemplary embodiments, the container 100 can be surrounded by asecond container 230. In some specific exemplary embodiments, the secondcontainer 230 can be made from polyethylene. In one aspect, the secondcontainer 230 can be capable of providing shatter resistant protectionfor glass bottles. In another aspect, the second container 230 can havean opening in the top.

In some exemplary embodiments, the container 110 can be made form aborosilicate glass material.

In some exemplary embodiments, a volume of the container 110 can rangefrom about 10 ml to about 5000 ml. In one aspect, a volume of thecontainer 110 can be about 10 ml, about 20 ml, about 30 ml, about 40 ml,about 50 ml, about 60 ml, about 70 ml, about 80 ml, about 90 ml, about100 ml, about 110 ml, about 120 ml, about 130 ml, about 140 ml, about150 ml, about 160 ml, about 170 ml, about 180 ml, about 190 ml, about200 ml, about 210 ml, about 220 ml, about 230 ml, about 240 ml, about250 ml, about 260 ml, about 270 ml, about 280 ml, about 290 ml, about300 ml, about 310 ml, about 320 ml, about 330 ml, about 340 ml, about350 ml, about 360 ml, about 370 ml, about 380 ml, about 390 ml, about400 ml, about 410 ml, about 420 ml, about 430 ml, about 440 ml, about450 ml, about 460 ml, about 470 ml, about 480 ml, about 490 ml, about1000 ml, about 1100 ml, about 1200 ml, about 1300 ml, about 1400 ml,about 1500 ml, about 1600 ml, about 1700 ml, about 1800 ml, about 1900ml, about 2000 ml, about 2500 ml, about 3000 ml, about 3500 ml, about4000 ml, about 4500 ml, or about 5000 ml.

In some exemplary embodiments, the container 110 can be a pressureresistant container.

In some exemplary embodiments, the container 110 can have a pressureresistance of at least about 0.5 bar gauge. In one aspect, the container110 can have a pressure resistance of at least about 0.5 bar gauge, atleast about 0.6 bar gauge, at least about 0.7 bar gauge, at least about0.8 bar gauge, at least about 0.9 bar gauge, at least about 1 bar gauge,at least about 1.1 bar gauge, at least about 1.2 bar gauge, at leastabout 1.3 bar gauge, at least about 1.4 bar gauge, at least about 1.5bar gauge, at least about 1.6 bar gauge, at least about 1.7 bar gauge,at least about 1.8 bar gauge, at least about 1.9 bar gauge, or at leastabout 2.0 bar gauge.

In some exemplary embodiments, the container 110 can be capable of beingfilled with at least one organic solvent. Non-limiting examples oforganic solvents include acetonitrile, propanol, isopropanol, water andmethanol.

In some exemplary embodiments, the container 110 can be capable of beingfilled with at least one acid. Non-limiting examples of acid includeacetic acid, propionic acid, and formic acid.

In some exemplary embodiments, the container capable 110 of being filledwith at least one base. Non-limiting examples of base include ammonia,diethylamine, triethylamine, N,N-diisopropylehtylamine (DIPEA), andpiperidine. In some exemplary embodiments, the container capable 110 ofbeing filled with at least one organic solvent and at least one acid.

In some exemplary embodiments, the container capable 110 of being filledwith at least one organic solvent and at least one base.

In some exemplary embodiments, the electrospray ionization source 100can be configured to allow a flow of a sheath gas from the sheath gasinlet line 120 through the container 110 capable of being filled with anorganic solvent and an additional chemical component into thedesolvation gas outlet line 130.

In some exemplary embodiments, the electrospray ionization source 100can be capable of providing an electrospray with a solvent flow rate ofgreater than about 5 μL/min. In one aspect, the electrospray ionizationsource 100 can be capable of providing an electrospray with a solventflow rate of greater than about 5 μL/min, greater than about 6 μL/min,greater than about 7 μL/min, greater than about 8 μL/min, greater thanabout 9 μL/min, greater than about 10 μL/min, greater than about 11μL/min, greater than about 12μL/min, greater than about 13 μL/min,greater than about 14 μL/min, greater than about 15μL/min, greater thanabout 16 μL/min, greater than about 17 μL/min, greater than about 18μL/min, greater than about 19 μL/min, greater than about 20 μL/min,greater than about 25 μL/min, greater than about 30 μL/min, greater thanabout 35 μL/min, greater than about 40 μL/min, greater than about 45μL/min, greater than about 50 μL/min, greater than about 55 μL/min,greater than about 60 μL/min, greater than about 65 μL/min, greater thanabout 70 μL/min, greater than about 75 μL/min, greater than about 80μL/min, greater than about 85 μL/min, greater than about 90 μL/min,greater than about 95 μL/min, greater than about 100 μL/min, greaterthan about 110 μL/min, greater than about 120 μL/min, greater than about130 μL/min, greater than about 140 μL/min, greater than about 150μL/min, greater than about 160 μL/min, greater than about 170 μL/min,greater than about 180 μL/min, greater than about 190 μL/min, greaterthan about 200 μL/min, greater than about 225 μL/min, greater than about250 μL/min, greater than about 275 μL/min, greater than about 300μL/min, greater than about 325 μL/min, greater than about 350 μL/min,greater than about 375 μL/min, greater than about 700 μL/min, greaterthan about 425 μL/min, greater than about 450 μL/min, or greater thanabout 500 μL/min.

In some exemplary embodiments, the mass spectrometer 30 can be capableof identifying components of the protein to characterize the protein.

In some exemplary embodiments, the mass spectrometer 30 can be a tandemmass spectrometer.

In some exemplary embodiments, the mass spectrometer 30 can be a tandemin time mass spectrometer.

In some exemplary embodiments, the mass spectrometer 30 can be a tandemin space mass spectrometer.

In some exemplary embodiments, the mass spectrometer 30 can be a hybridwherein tandem-in-time analyzer can be coupled in space or withtandem-in-space analyzer.

In some exemplary embodiments, the mass spectrometer 30 can be aquadrupole-Orbitrap hybrid mass spectrometer. The quadrupole-Orbitraphybrid mass spectrometer can be Q Exactive™ Focus HybridQuadrupole-Orbitrap™ Mass Spectrometer, Q Exactive™ Plus HybridQuadrupole-Orbitrap™ Mass Spectrometer, Q Exactive™ BioPharma Platform,Q Exactive™ UHMR Hybrid Quadrupole-Orbitrap™ Mass Spectrometer, QExactive™ HF Hybrid Quadrupole-Orbitrap™ Mass Spectrometer, Q Exactive™HF-X Hybrid Quadrupole-Orbitrap™ Mass Spectrometer, and Q Exactive™Hybrid Quadrupole-Orbitrap™ Mass Spectrometer.

In some exemplary embodiments, the mass spectrometer 30 can be aQIT-FTICR.

In some exemplary embodiments, the mass spectrometer 30 can be aQq-FTICR.

In some exemplary embodiments, the method of characterizing a protein ina sample can comprise detecting or quantifying the protein in thesample.

In one exemplary embodiment, the protein can include an antibody,bispecific antibody, antibody fragment or a multispecific antibody.

In some exemplary embodiments, the protein can be a therapeuticantibody.

In some exemplary embodiments, the protein can be an immunoglobulinprotein.

In one exemplary embodiment, immunoglobulin protein can be IgG1.

In one exemplary embodiment, immunoglobulin protein can be IgG4.

In some exemplary embodiments, the protein can be a bispecific antibody.

In some exemplary embodiments, the protein can be an antibody fragmentformed on digestion of the antibody.

In one exemplary embodiment, the protein can be a protein variant.

In one exemplary embodiment, the protein can be a post-translationallymodified protein.

In one exemplary embodiment, the post-translationally modified proteincan be a formed by cleavage, N-terminal extensions, protein degradation,acylation of the N-terminus, biotinylation, amidation of the C-terminal,oxidation, glycosylation, iodination, covalent attachment of prostheticgroups, acetylation, alkylation, methylation, adenylation,ADP-ribosylation, covalent cross links within, or between, polypeptidechains, sulfonation, prenylation, Vitamin C dependent modifications,Vitamin K dependent modification, glutamylation, glycylation,glycosylation, deglycosylation, isoprenylation, lipoylation,phosphopantetheinylation, phosphorylation, sulfation, citrullination,deamidation, formation of disulfide bridges, proteolytic cleavage,ISGylation, SUMOylation or ubiquitination (covalent linkage to theprotein ubiquitin).

In one exemplary embodiment, the post-translationally modified proteincan be formed on oxidation of a protein.

In another exemplary embodiment, the degradation product can include apost-translation modification of a therapeutic protein.

In another exemplary embodiment, the protein can be a degradationproduct of a protein.

In yet another exemplary embodiment, the protein can be an impurityfound in a biopharmaceutical product.

In another exemplary embodiment, the protein can be an impurity foundduring the manufacture of the biopharmaceutical product.

In some exemplary embodiments, the protein can be a protein with a pI inthe range of about 4.5 to about 9.0. In one aspect, the protein can be aprotein with a pI of about 4.5, about 5.0, about 5.5, about 5.6, about5.7, about 5.8, about 5.9, about 6.0, about 6.1 about 6.2, about 6.3,about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about7.0, about 7.1 about 7.2, about 7.3, about 7.4, about 7.5, about 7.6,about 7.7, about 7.8, about 7.9, about 8.0, about 8.1 about 8.2, about8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9,or about 9.0.

In some exemplary embodiments, the protein can be a product-relatedimpurity. The product related impurity can be molecular variants,precursors, degradation products, fragmented protein, digested product,aggregates, post-translational modification form or combinationsthereof.

In some specific exemplary embodiments, the protein can be aprocess-related impurity. The process-related impurity can includeimpurities derived from the manufacturing process, e.g., nucleic acidsand host cell proteins, antibiotics, serum, other media components,enzymes, chemical and biochemical processing reagents, inorganic salts,solvents, carriers, ligands, and other leachables used in themanufacturing process.

In some specific exemplary embodiments, the protein can be an impurity.In one exemplary embodiment, the number of impurities in the sample canbe at least two.

In some exemplary embodiments, the mobile phase used to elute theprotein can be a mobile phase that can be compatible with a massspectrometer.

In some exemplary embodiments, the mobile phase used in the liquidchromatography device can include water, acetonitrile, trifluoroaceticacid, formic acid, or combination thereof.

In some exemplary embodiments, the mobile phase can have a flow rate ofabout 0.1 ml/min to about 0.4 ml/min in the liquid chromatographydevice. In one aspect, the flow rate of the mobile phase in the liquidchromatography device can be about 0.1 ml/min, about 0.15 ml/min, about0.20 ml/min, about 0.25 ml/min, about 0.30 ml/min, about 0.35 ml/min, orabout 0.4 ml/min.

It is understood that the system is not limited to any of the aforesaidprotein, impurity, mobile phase, mass spectrometer, organic solvent,acid, base, or chromatographic column.

The consecutive labeling of method steps as provided herein with numbersand/or letters is not meant to limit the method or any embodimentsthereof to the particular indicated order.

Various publications, including patents, patent applications, publishedpatent applications, accession numbers, technical articles and scholarlyarticles are cited throughout the specification. Each of these citedreferences is incorporated by reference, in its entirety and for allpurposes, herein.

The disclosure will be more fully understood by reference to thefollowing Examples, which are provided to describe the disclosure ingreater detail. They are intended to illustrate and should not beconstrued as limiting the scope of the disclosure.

EXAMPLES

Materials.

Deionized water was provided by a Milli-Q integral water purificationsystem installed with a MilliPak Express 20 filter (Millipore Sigma,Burlinton, Mass.). NIST Monoclonal Antibody Reference Material 8671(NISTmAb, humanized IgG1K monoclonal antibody) was purchased fromNational Institute of Standards and Technology (Gaithersburg, Md.).Rapid Peptide N-glycosidase F (Rapid PNGase F) with 5× Rapid PNGase Fbuffer was purchased from New England Biolabs Inc. (Ipswich, Mass.).TCEP-HCl (tris(2-carboxyethyl) phosphine hydrochloride), Tris-HCl pH 7.5(UltraPure), trifluoroacetic acid (LC-MS grade), formic acid (LC-MSgrade), and acetonitrile (Optima LC/MS grade) were purchased from ThermoFisher Scientific (Waltham, Mass.), trypsin (sequencing grade) waspurchased from Promega (Madison, Mich.). Iodoacetamide, propionic acidand acetic acid were purchased from Sigma Aldrich, Co. (St. Louis, Mo.).2-propanol (HPLC grade) was purchased from VWR International, LLC(Radnor, Pa.).

Safety Considerations.

For desolvation gas modified experiments, the sheath gas was set to 15arbitrary units. A higher setting might be possible, but the pressurewithin the bottle should be measured and made sure not to exceed thepressure rating of the bottle. A pressure resistant bottle (e.g., Duranpressure plus bottle, pressure rating: +1.5 bar) was high recommendedfor this application. A secondary container was also required for thissetup to prevent possible acid or base spills.

Example 1

1.1 Sample Preparation.

NISTmAb stock sample (100 μg) was diluted into 5 mM acetic acid andreduced with 5 mM TCEP-HCl at 80° C. for 10 min. After adjusting the pHto 7.5 using 1 M Tris-HCl (pH 7.5), 10 mM iodoacetamide and 5 μg oftrypsin (E/S ratio at 1:20) were added, and the sample was incubated at37° C. for 3 hours. Finally, the solution was acidified by 1% TFA toquench the digestion.

1.2 LC-MS Analysis.

For peptide mapping analysis of NISTmAb, aliquots (2 μg) of digests wereseparated using an ACQUITY UPLC Peptide BEH C18 Column (130 Å, 1.7 μm,2.1 mm×150 mm) (Waters, Milford, Mass.) for online LC-MS/MS analysis ona Q-Exactive mass spectrometer. For the separation, the mobile phase Awas 0.1% FA (v/v) in water, and mobile phase B was 0.1% FA inacetonitrile (ACN). Detailed LC gradient and MS parameters are includedin the Tables 1 and 2 respectively.

TABLE 1 LC gradient for peptide mapping analysis Mobile A: 0.05%Trifluoroacetic Acid in Water or 0.1% Formic Phase Acid in Water B:0.045% Trifluoroacetic Acid in Acetonitrile or 0.1% Formic Acid inAcetonitrile Column Waters ACQUITY UPLC Peptide BEH C18 130 Å, 1.7 μm,2.1 mm × 150 mm column Column 40° C. Temperature Gradient Time (min)Flow (μL/min) % A % B 0.0 0.250 99.9 0.1 5.0 0.250 99.9 0.1 80.00 0.25065.0 35.0 80.10 0.250 10.0 90.0 90.00 0.250 10.0 90.0 91.00 0.250 99.90.1 105.00 0.250 99.9 0.1

TABLE 2 MS parameters for peptide mapping analysis Control Desolvationgas modified MS parameters Experiment method (w/PA/IPA) Probe heatertemperature 250 250  [° C.] Source voltage [kV] 3.8   3.8 Capillarytemperature [° C.] 320 320  S-lens RF level 60 60 Sheath gas 40  15^(a)Aux gas 10 10 Sweep gas 0  0 Scan range [m/z] 300-2000 300-2000 ^(a)thesheath gas setting was reduced to 15 arbitrary units to reduce thepressure within the solvent bottle.

1.3 Results

FIG. 3 shows the base peak chromatograms (BPCs) from the LC-MS analysisof the tryptic digests of NISTmAb using FA as a mobile phase with a BEHC18 column.

Example 2

2.1 Sample Preparation.

Sample preparation was carried out as described in 1.1

2.2 LC-MS Analysis.

For peptide mapping analysis of NISTmAb, aliquots (2 μg) of digests wereseparated using an ACQUITY UPLC Peptide BEH C18 Column (130 Å, 1.7 μm,2.1 mm×150 mm) (Waters, Milford, Mass.) for online LC-MS/MS analysis ona Q-Exactive mass spectrometer. For the separation, mobile phase A was0.05% TFA (v/v) in water, and mobile phase B was 0.045% TFA (v/v) inACN. The LC gradient and MS parameters implemented are included in theTables 1 and 2 respectively.

2.3 Results

FIG. 4 shows the base peak chromatograms (BPCs) from the LC-MS analysisof the tryptic digests of NISTmAb using TFA as a mobile phase with a BEHC18 column. On comparing the BPCs in FIGS. 3 and 4, it is evident thatthe separation and retention of tryptic peptides on the C18 column weresignificantly different using either FA or TFA as mobile phase modifier.In general, TFA outperforms FA from the chromatographic performanceperspective, exhibiting better retention of hydrophilic peptides, betterpeak shapes and overall higher peak capacity. For example, a smalltryptic peptide EYK (P1, FIG. 4) was not retained on the C18 column whenFA was used, whereas it is retained on the same column when TFA wasapplied. This feature might improve the sequence coverage of proteinbiopharmaceuticals from the peptide mapping analysis. On the other hand,TFA-based analysis exhibited a significant loss in MS sensitivitycomparing to FA-based analysis.

Example 3

3.1 Sample Preparation.

Sample preparation was carried out as described in 1.1

3.2 LC-MS Analysis.

LS-MS analysis was carried out as described in 2.2.

3.3 Modification of the Desolvation Gas

The sheath gas flow from a Q-Exactive mass spectrometer was redirectedto a Duran pressure plus bottle (SCHOTT North America, Inc., Elmsford,N.Y.) through a Canary-Safe Cap (Analytical Sales and Services, Inc.,Flander, N.J.) using ⅛″ TEFLON tubing (FIG. 1). The outgoing tubing fromthe bottle was then connected back to the HESI-II probe in a ThermoScientific Ion Max ion source. For peptide mapping analysis, 150 mL ofpropionic acid (PA) and 50 mL of isopropanol (IPA) were mixed andtransferred into the bottle. The bottle containing PA and IPA was thenplaced into a polyethylene secondary container (BEL-ART acid/solventbottle carrier, Wayne, N.J.) with a 16 mm opening in the top forinsertion of tubing. To disable the modification, the sheath gas tubingwas directly connected to the HESI-II probe without passing through thedevice.

3.4 Results

FIG. 5 shows the base peak chromatograms (BPCs) from the LC-MS analysisof the tryptic digests of NISTmAb using trifluoroacetic acid as mobilephase with a BEH C18 column with desolvation modification enabled. Oncomparing the BPCs in FIG. 5 (TFA-containing mobile phase, desolvationgas modification enabled) with FIG. 3 (FA-containing mobile phase,desolvation gas modification disabled) and FIG. 4 (TFA-containing mobilephase, desolvation gas modification disabled), it is evident thatmodification of the desolvation gas with acid vapor from PA and IPA ledto a significant increase in MS sensitivity for TFA-based analysis.

As demonstrated by six representative tryptic peptides of different sizeand retention time (Table 3 below), 4.9-5.8 folds of increase in MSsensitivity were readily achieved by this approach, regardless of thepeptide size and mobile phase composition. In addition, a subtleincrease in charge states were also consistently observed for manytryptic peptides after the desolvation gas modification. This featuremight lead to improved spectral quality of tandem MS due to moreefficient fragmentation of highly charged species, and subsequentlyresulting in more confident identification. Overall, by modifying thedesolvation gas with acid vapor using the developed device, asignificantly more sensitive peptide mapping method can be readilyachieved. The gained MS sensitivity is of great value to characterizelow-abundance attributes present in protein biopharmaceuticals, such asPTMs and sequence variants.

TABLE 3 Comparison of the MS intensities and averaged charge states ofsix representative tryptic peptides from NISTmAb analyzed under thethree conditions. MS intensities (ion counts)/averaged charge states TFATFA Increase Tryptic peptides FA control w/PA/IPA (folds) P1: EYK NA9.7E+07/ 4.3E+08/ NA z = 1.00 z = 1.00 P2: VDNALQSGNSQESVTEQDSK 3.4E+09/4.9E+08/ 2.4E+09/ 4.9 z = 2.65 z = 2.19 z = 2.19 P3: VYACEVTHQGLSSPVTK8.5E+09/ 1.05E+09/ 6.0E+09/ 5.7 z = 2.81 z = 2.21 z = 2.57 P4:STSGGTAALGCLVK 8.3E+09/ 1.2E+09/ 5.6E+09/ 4.9 z = 1.96 z = 1.88 z = 1.89P5: TTPPVLDSDGSFFLYSK 9.4E+09/ 1.3E+09/ 7.7E+09/ 5.8 z = 2.16 z = 1.98 z= 2.00 P6: DYFPEPVTVSWNSGALTSGVHTFPA 4.5E+09/ 7.1E+08/ 4.0E+09/ 5.7VLQSSGLYSLSSVVTVPSSSLGTQTY z = 5.47 z = 4.81 z = 5.05 ICNVNHKPSNTK

Example 4

4.1 Sample Preparation.

Initial sample preparation was carried out as described in 1.1. However,to evaluate if the new approach can be applied to continuous analysisand if the improved sensitivity can be maintained over a long period oftime for peptide mapping analysis, NISTmAb digests were repeatedlyperformed over two consecutive days.

4.2 LC-MS Analysis.

LS-MS analysis was carried out as described in 2.2.

4.3 Modification of the Desolvation Gas

The desolvation gas modification was carried out as described in 3.3.

4.4 Results

Again, after enabling the desolvation gas modification with acid vaporas described in 3.3, the increase in MS sensitivity, comparing to thecontrol method (as described in example 3), was immediately achieved andmaintained for at least 37 consecutive runs (˜3800 min) as tested (SeeFIG. 6). The consumption of the PA and IPA mixture, under the appliedexperimental conditions, was estimated to be 1 mL per hour. As a result,the developed approach is highly suitable for routine peptide mappinganalysis of protein biopharmaceuticals, where continuous analysis oflarge sample sets might be frequently required.

Example 5

Examples 1-4 illustrate the results of using the desolvate gas approachfor peptide mapping analysis. To evaluate if the same approach can beutilized to improve the intact mass analysis for proteinbiopharmaceuticals via charge reduction, an experiment using thedesolvation gas approach with weak base was conducted.

5.1 Sample Preparation

10 mM TCEP-HCl was added to diluted NISTmAb solution (1 μg/μL), and thesample was incubated at 50° C. for 30 minutes. To remove the N-glycanspresent on reagent protein X, the protein sample was first diluted to0.25 μg/μL using 5×Rapid PNGase F buffer (containing reducing agent),and the solution was then incubated at 80° C. for 10 min. Subsequently,Rapid PNGase F was added to the solution, and the sample was incubatedat 50° C. for 30 minutes.

5.2 RPLC Analysis

For intact mass analysis of reduced NISTmAb and reagent protein X.Aliquots (4 μg) of each sample were separated using a BioResolve RP mAbPolyphenyl column (50 mm×2.1 mm, 2.7 μm) (Waters, Milford, Mass.) foronline LC-MS analysis on a Q-Exactive mass spectrometer. The detailed LCgradient and MS parameters are included in the Tables 4 and 5,respectively.

TABLE 4 LC gradient for intact mapping analysis Mobile A: 0.1% FormicAcid in Water Phase B: 0.1% Formic Acid in Acetonitrile Column WatersBioResolve RP mAb Polyphenyl, 450 Å, 2.7 μm, 2.1 × 50 mm column Column80° C. Temperature Gradient Time (min) Flow (μL/min) % A % B 0.0 0.25085.0 15.0 10.0 0.250 45.0 55.0 10.15 0.250 20.0 80.0 10.65 0.250 20.080.0 10.80 0.250 85.0 15.0 12.50 0.250 85.0 15.0

TABLE 5 MS parameters for intact mass analysis of reduced NISTmAb andreagent protein X Control Desolvation gas modified MS parametersExperiment method w/1% TEA in ACN Probe heater temperature 250 250  [°C.] Source voltage [kV] 3.5   3.5 Capillary temperature [° C.] 350 350 S-lens RF level 60 60 Sheath gas 20  15^(a) Aux gas 10 10 Sweep gas 0  0Scan range [m/z] 800-4000 1500-5500^(b) SID [eV] 0 75 ^(a)the sheath gassetting was reduced to 15 arbitrary units to reduce the pressure withinthe solvent bottle. ^(b)the scan region was changed due to the chargereduction.

5.3 Modification of the Desolvation Gas

The sheath gas flow from a Q-Exactive mass spectrometer was redirectedto a Duran pressure plus bottle (SCHOTT North America, Inc., Elmsford,N.Y.) through a Canary-Safe Cap (Analytical Sales and Services, Inc.,Flander, N.J.) using ⅛″ TEFLON tubing (See FIG. 1). The outgoing tubingfrom the bottle was then connected back to the HESI-II probe in a ThermoScientific Ion Max ion source. For intact mass analysis, 1%triethylamine (TEA) (v/v) in 200 mL of acetonitrile was transferred intothe bottle. The bottle containing concentrated acid or base was thenplaced into a polyethylene secondary container (BEL-ART acid/solventbottle carrier, Wayne, N.J.) with a 16 mm opening in the top forinsertion of tubing. To disable the modification, the sheath gas tubingwas directly connected to the HESI-II probe without passing through thedevice.

5.4 Results

To evaluate if similar improvements can be achieved via desolvation gasmodification, reduced NISTmAb was used as a testing article.Triethylamine (TEA) was diluted to 1% (v/v) using acetonitrile (ACN) andused to deliver the base vapor into desolvation gas. Afterchromatographic separation on a reversed-phase (RP) polyphenol column,the MS spectra of reduced heavy chain of NISTmAb acquired from a controlmethod and from the desolvation gas modified method were both shown inFIG. 7 (control method) and FIG. 8 (charge reduction method). Afterenabling the modification, a significant charge reduction on NISTmAbheavy chain, from charge states+23 to +55 to charge states+14 to +28,was achieved. Close examination of charge state+39 from the controlmethod revealed a high level of background noise, which was likelyattributed to the decay of the highly charged heavy chain species duringthe MS analysis, such as neutral losses from water (−18 Da), ammonia(−17 Da) and carboxylic acid (−44 Da) as well as protein backbonefragmentation.

On the contrary, the zoom-in view of charge state+21 from the modifiedmethod exhibited greatly improved signal to noise ratio, thus allowingthe identification of several low abundance glycoforms that are notdetected in the control method. The deconvoluted mass spectrum (See FIG.9) and mass analysis (Table 6) further demonstrated that the improvedmethod could accurately identify glycoforms present at as low as 0.3% onthe heavy chain of NISTmAb. The improvement in spectral quality is notonly attributed to greatly simplified spectrum due to less crowdedcharge state envelopes, but more importantly is a result of stabilizedprotein analytes. This is consistent with the well-established knowledgethat the collision energy associated with a protein ion during MSanalysis is proportional to its charge state. Specifically, lower chargestates decay slower than the higher charge states of the same analyteduring the analysis within orbitrap. Notably, efforts to mitigate theextensive decay of highly charged heavy chain species from the controlmethod were not successful, even after completely removing thesource-induced dissociation (SID) energy. On the contrary, high levelsof SID energy (75 eV used in this experiment) can be readily toleratedfrom the charge reduction method without noticeable decay orfragmentation of heavy chain, which further improves the spectralquality via more efficient desolvation and adduct removal. Similarimprovement in spectral quality was also achieved for the reduced lightchain of NISTmAb (See FIG. 10). It is worth noting that application ofcharge reduction strategy to improve MS spectral quality is most helpfulfor fully denatured and reduced proteins, as they are often highlycharged (due to increased protein surface areas (Kaltashov and Mohimen.Analytical chemistry 2005, 77, 5370-5379)) during ESI-MS analysis andare most susceptible to undesired fragmentation and decay.

TABLE 6 Theoretical Observed Mass of Glycosylated Average GlycosylatedHeavy Chain Glycan Mass % Peak Heavy Chain Mass Glycan (Da) Area (Da)(Da) A1 1096.0 0.4% 50557.5 50557.3 M5 1217.1 0.5% 50678.6 50676.9 FA11242.1 2.1% 50703.6 50703.3 FA1G1 1404.3 2.9% 50865.8 50864.5 FA2 1445.339.3% 50906.8 50906.5 FA2G1 1607.5 40.4% 51069.0 51068.6 FA2G2 1769.611.1% 51231.1 51230.8 FA2G2 + Hex 1931.7 2.3% 51393.2 51393.1 FA2G2 +2Hex 2093.9 0.7% 51555.4 51555.8 FA2G2 + 2239.0 0.3% 51700.5 51699.8Hex + Gc

Example 6

In addition, the developed charge reduction method, as achieved bymodifying the desolvation gas with TEA, can also be utilized to tacklehigh mass heterogeneity present in complex protein samples. Those mightinclude various protein reagents that are critically important tosupport different assays during the development of proteinbiopharmaceuticals. Using intact mass analysis to confirm the identitiesof those protein reagents, no matter if they are obtained fromcommercial sources or produced in-house is frequently required tosupport the subsequent studies. Some protein reagents are highlyheterogeneous in molecular weight due to extensive glycosylation, whichcan present significant challenges for routine intact mass method. Todemonstrate the utility of the developed charge reduction method, acomplex reagent protein (Protein X) with high mass heterogeneity wasused as a testing article.

6.1 Sample Preparation.

Prior to the analysis, Protein X was first treated with PNGase F underboth denaturing and reducing conditions, in order to remove the massheterogeneity introduced by the presence of N-glycans. As shown in FIG.11, even after the PNGase F treatment, Protein X exhibited a highlyconvoluted MS spectrum that cannot be deciphered using a regular intactmass method. This was likely attributed to the overlapping chargeenvelopes from different co-eluting mass forms of this molecule at lowm/z region.

6.3 Modification of the Desolvation Gas.

The desolvation gas modification was carried out as described in 5.3.

6.4 Results

In contrast to results obtained for example 6.1, after enabling thedesolvation gas modification with TEA, the MS signal of the same samplewas immediately shifted to high m/z region and exhibiting much betterresolved charge states. Subsequently, the deconvoluted spectrum (SeeFIG. 12) clearly revealed that this protein was extensively modified byO-glycans with possibly 10 different glycosylation sites and fourdifferent O-glycan forms. Overall, nearly thirty five different massspecies from this protein were confidently identified and assigned. Itis worth noting that the dramatically improved spectral quality mightalso be partly attributed to more stabilized protein ions after chargereduction, as the polysaccharide moiety on this molecule could be labileunder regular ESI-MS conditions.

Finally, for some other LC-MS based intact mass methods, where the useof TFA is inevitable to ensure chromatographic performance, thedeveloped approach was also tested to counteract TFA ion suppression atprotein level using PA/IPA modified desolvation gas. For example,hyphenation of size exclusion chromatography (SEC) to MS using mobilephases containing acetonitrile, TFA, and formic acid has been used forreduced mAb analysis. (Liu et al. Journal of the American Society forMass Spectrometry 2009, 20, 2258-2264.) Hyphenation of hydrophilicinteraction chromatography to MS using TFA-containing mobile phases hasbeen used to study the low molecular weight impurities in mAb samples(Wang et al. Journal of pharmaceutical and biomedical analysis 2018,154, 468-475). In both methods, significant improvement in MSsensitivity can be achieved for many mAb fragments (e.g. heavy chain,light chain and smaller fragments) using PA/IPA modified desolvationgas. However, the MS sensitivity of intact mAb (˜150 kDa) did notimprove as a result of this modification. This finding is alsoconsistent with the previous study (Apffel et al. Journal ofchromatography. A 1995, 712, 177-190) which hypothesizes that a largerprotein might accommodate a greater number of TFA anions, which cannotbe effectively replaced by PA during the ESI process.

Thus, an effective approach to improve the data quality from bothpeptide mapping analysis and intact mass analysis via desolvation gasmodification using a simple device is demonstrated. By using PA/IPAmodified desolvation gas, the TFA ion suppression from a typical peptidemapping method can be effectively mitigated, and thus leading tosignificantly improved MS sensitivity. The developed approach can beeasily implemented without changing the LC method and is capable ofcontinuous analysis of large sample sets, making it particularlysuitable for routine characterization of protein biopharmaceuticals. Byusing TEA modified desolvation gas, the new approach could also beutilized to improve the intact mass analysis of proteins via chargereduction. Significant improvement in spectral quality not only allowsthe detection of minor mass forms otherwise buried in noise, but alsoenables the mass measurement of highly heterogeneous proteins. Finally,with the ever-increasing role played by LC-MS technique in proteinbiopharmaceutical characterization, the developed approach can make adeeper and broader contribution by serving as a low-cost and practicalsolution to improve the analytical capability and better support thedrug development.

What is claimed is:
 1. An electrospray ionization source, comprising: acontainer having a cap, wherein the cap has an inlet line port and anoutlet line port, wherein the container comprises an organic solvent anda base; a sheath gas inlet line for providing a sheath gas to the inletline port; and a modified desolvation gas outlet line capable ofconnecting the outlet line port to a sheath gas inlet of an electrosprayionization probe, wherein the electrospray ionization probe is operatedin positive ion mode, wherein the organic solvent and base are capableof reducing a charge state of an analyte ion.
 2. The electrosprayionization source of claim 1, wherein the organic solvent isacetonitrile.
 3. The electrospray ionization source of claim 1, whereinthe base is triethylamine.
 4. The electrospray ionization source ofclaim 1, wherein the electrospray ionization probe comprises anauxiliary gas inlet.
 5. The electrospray ionization source of claim 4,wherein the auxiliary gas inlet is supplied with an auxiliary gas. 6.The electrospray ionization source of claim 1, wherein the electrosprayionization probe comprises an electrospray emitter needle, a sheath gasflow plumbing, and an auxiliary gas flow plumbing.
 7. The electrosprayionization source of claim 6, wherein the electrospray ionization probeis configured to direct flow in the sheath gas flow plumbing coaxiallyto the electrospray emitter needle.
 8. The electrospray ionizationsource of claim 6, wherein the electrospray ionization probe isconfigured to direct the flow in the auxiliary gas flow plumbingcoaxially to the electrospray emitter needle.
 9. The electrosprayionization source of claim 1, wherein the sheath gas inlet line ispartially inserted into to the inlet line port.
 10. The electrosprayionization source of claim 1, wherein the modified desolvation gasoutlet line is partially inserted into to the outlet line port.
 11. Theelectrospray ionization source of claim 1, wherein the sheath gas flowsfrom the sheath gas inlet line through the container containing anorganic solvent into the desolvation gas outlet line.
 12. Theelectrospray ionization source of claim 1, wherein the container issurrounded by a second container.
 13. The electrospray ionization sourceof claim 1, wherein the electrospray ionization source is capable ofbeing connected to a liquid chromatographic system.
 14. A method ofcharacterizing a protein in a sample, comprising: supplying the sampleto an inlet of an electrospray ionization source; wherein theelectrospray ionization source comprises a container having a cap,wherein the cap has an inlet line port and an outlet line port, whereinthe container comprises an organic solvent and a base; a sheath gasinlet line for providing a sheath gas to the inlet line port; and amodified desolvation gas outlet line capable of connecting the outletline port to a sheath gas inlet of an electrospray ionization probe;generating ions of components of the protein in the sample at an outletof the electrospray ionization source; and analyzing the ions using amass spectrometer to identify the components of the protein tocharacterize the protein, wherein the electrospray ionization probe isoperated in positive ion mode, and wherein the organic solvent and basereduce a charge state of the ions.
 15. The method of claim 14, whereincharacterizing the protein comprises conducting an intact mass analysis.16. The method of claim 14, wherein characterizing the protein comprisesconducting a peptide mapping analysis.
 17. The method of claim 14,wherein the electrospray ionization source is provides an electrospraywith a solvent flow rate of greater than about 5 μL/min.
 18. A liquidchromatography mass spectrometry system, comprising: a liquidchromatography device configured to direct a flow of a sample to anelectrospray ionization source; wherein the electrospray ionizationsource comprises a container having a cap, wherein the cap has an inletline port and an outlet line port, wherein the container comprises anorganic solvent and a base; a sheath gas inlet line for providing asheath gas to the inlet line port; and a modified desolvation gas outletline capable of connecting the outlet line port to a sheath gas inlet ofan electrospray ionization probe; and wherein the electrosprayionization source is configured to charge and desolvate the sample toform ions of components of the sample; and a mass spectrometry deviceconfigured to receive the ions and characterize mass to charge ratio ofthe ions, wherein the electrospray ionization probe is operated inpositive ion mode, and wherein the organic solvent and base are capableof reducing a charge state of the ions.